Large Molecular Fragments Research Articles

Enhanced Performance Electrochemical Biosensor for Detection of Prostate Cancer Biomarker PCA3 Using Specific Aptamer

In the quest for the development of accurate, reliable, and cost-effective biosensing technology for early diagnostics of prostate cancer, we describe here an electrochemical biosensor combining a simple transducing method of differential pulse voltammetry (DPV) with an RNA-based aptamer labelled with a methylene blue redox group acting as a highly specific bioreceptor to the prostate cancer biomarker PCA3. A series of DPV measurements on screen-printed gold electrodes is functionalised with a redox-labelled aptamer in solutions (either buffer or synthetic urine) containing PCA3 in a wide range of concentrations from 0.1 picomolar (pM) to 10 nanomolar (nM). In these measurements, the current peak values correlate with the concentration of PCA3 and yield a low detection limit (LDL) of 0.1 pM. Furthermore, the binding kinetics study revealed the high affinity of the aptamer to the target PCA3 with the affinity constants KD of about 3.0 × 10−8 molar. In addition, the AFM study showed the increase in the molecular layer roughness caused by the binding of PCA3, which is a large RNA molecular fragment.

Molecular Dynamics Simulations of the Thermal Decomposition of 3,4-Bis(3-nitrofurazan-4-yl)furoxan.

When stimulated, for example, by a high temperature, the physical and chemical properties of energetic materials (EMs) may change, and, in turn, their overall performance is affected. Therefore, thermal stability is crucial for EMs, especially the thermal dynamic behavior. In the past decade, significant efforts have been made to study the thermal dynamic behavior of 3,4-bis(3-nitrofurazan-4-yl)furoxan (DNTF), one of the new high-energy-density materials (HEDMs). However, the thermal decomposition mechanism of DNTF is still not specific or comprehensive. In this study, the self-consistent-charge density-functional tight-binding method was combined with molecular dynamics (MD) simulations to reveal the differences in the thermal decomposition of DNTF under four heating conditions. The O–N (O) bond would fracture first during DNTF initial thermal decomposition at medium and low temperatures, thus triggering the cracking of the whole structure. At 2000 and 2500 K, NO2 loss on outer ring I is the fastest initial thermal decomposition pathway, and it determines that the decomposition mechanism is different from that of a medium-low temperature. NO2 is found to be the most active intermediate product; large molecular fragments, such as C2N2O, are found for the first time. Hopefully, these results could provide some insights into the decomposition mechanism of new HEDMs.

The characteristic of agarose extraction from dimethyl sulfide and isopropanol

Introduction. This study aimed to obtain agarose product with market standard quality which could be applied for electrophoresis. Methods. The material used in agarose extraction process was dried agar – agar bar extracted using dimethyl sulfide and soaked in isopropanol solution with 85%, 90%, and 95% of isopropanol concentration. The parameter which observed in this study was consisted of yield, water content, sulfate content, ash content, gel strength and electrophoresis gel. Results. The average score of agarose yield using different concentrations of isopropanol (IPA) was around 46.76% - 57.53% with the lowest yield of 46.76% and the highest yield of 57.53%. Meanwhile the average water content ranged from 18.32% to 18.64%. The average ash content was around 0.15% - 0.16% and the average sulfate content was 0.14% - 0.15%. The average strength of a 1% gel prepared in isopropanol was 1170 – 1183.33 gr/cm2. Best IPA concentration to soak agarose extract was on 95% concentration, with market standard characteristic as 57.54% of yield, 18.35% of water content, 0.14% of sulfate content, 0.15% of ash content and 1183.33 of gel strength. Conclusions.The application of electrophoresis of agarose gel showed that the bands of large molecular DNA fragment could not separate. Partially hydrolyzed protein samples had higher protein content, lighter color, and lower degree of denaturation and better functional properties compared to the traditional protein isolates.

Release of large polycyclic aromatic hydrocarbons and fullerenes by cosmic rays from interstellar dust

Context. Top-down chemistry is believed to be responsible for the formation of the large molecular compounds such as the polycyclic aromatic hydrocarbon-like molecules and the fullerenes observed in the interstellar medium. The release of these large molecules from the parent grains remains an important issue to be investigated. Aims. Cosmic rays irradiate the dust grains during their journey in the interstellar medium. In this study we probe to what extent electronic sputtering and/or desorption processes induced by high-energy ion projectiles contribute to the creation of the large molecular component in space. Methods. Carbonaceous dust analogues were produced in an ethylene flame. The resulting soot nanoparticles generated under well-defined conditions were irradiated by swift heavy ions, and mass spectra of the ionic and neutral molecular fragments emitted shortly after the impact were monitored. Results. Large molecular fragments were detected, including neutral and ionic polycyclic aromatic hydrocarbons containing up to several tens of carbon atoms, as well as ionic fullerenes. Although the absolute efficiencies were not obtained, these experiments provide a proof of principle of a top-down scenario involving interaction processes of interstellar dust with high-energy projectiles yielding large molecular compounds observed in space.

Emergent functional behaviour of humic substances perceived as complex labile aggregates of small organic molecules and oligomers

Environmental contextThe correlation of physicochemical characteristics of humic substances with their function is crucial to our understanding of how environmental pollutants interact with humic substances. We have developed an approach that models emergent functions of fulvic and humic acids depending on sample characteristics. The results will be useful for predicting the sequestration of organic contaminants in soil under various conditions. AbstractThe structural organisation of humic substances (HS) has been a central question of earth sciences for several decades. The latest experimental results have led to the recognition of HS as complex mixtures of small molecules and oligomers. We investigate the correlation between the chemical composition of HS, perceived as labile aggregates, and the emergent functions. Computational modelling was used to help to understand the processes and mechanisms on the molecular scale that occur in different fractions of the HS, fulvic acids (FA) and humic acids (HA), as they interact with metal ions and organic pollutants. The importance of non-covalent interactions in the emergent functions of HS is highlighted. H-bonding, hydrophilic/hydrophobic surface areas and π-stacking interactions play a significant role in aggregation processes as well as in the sorption of environmental pollutants. In a highly hydrophilic system with small molecules (the SRFA-22 model), H-bonding is the main force that drives the aggregation process. However, in a highly aromatic and hydrophobic model with larger molecular fragments (SRHA-6), hydrophobic and π-stacking interactions dominate in the aggregation process. The chemical properties of contaminants significantly affect their mechanisms of sorption by HS. The interaction of a polar pollutant, phenol, with HS occurs through H-bonding, whereas non-polar benzene interacts through hydrophobic and π-stacking interactions. The non-polar pollutant results in a much stronger sorption by HS and causes an additional structural rearrangement of the aggregates, which make it more stable in the environment.

Hydrothermal degradation of hemicelluloses from triploid poplar in hot compressed water at 180–340 °C

Hemicelluloses isolated from triploid poplar were subjected to hydrothermal degradation at 180–340 °C. The detailed physicochemical characteristics of hydrothermal degradation products including monosaccharide and XOS, molecular weight, inhibitory and organic compounds were detected by HPAEC, GPC, HPLC, and GC–MS, respectively. An increase in temperature led to a significant decrease in molecular weight and an increase in C2–C5 organic alcohols and acids. More importantly, besides two well-known routes that hemicelluloses are hydrolyzed to monosaccharide, followed by further degraded to furfural, a series of reaction routes was proposed to clarify and explain the mechanisms of the hydrothermal degradation of hemicelluloses from triploid poplar, which involved: (i) a predominant route of fragmentation; (ii) side reactions such as oxidation, dehydration, isomerization/rearrangement, and self-lactonization; (iii) larger molecular fragments from sugars by deoxydation or/and oxidation, followed by self-lactonization; and (iv) very few extensive re-polymerization including esterification and condensation among hemicelluloses and lignin fragments.

Through-space and through-bridge interactions in the correlation analysis of chemical bonds

The formalism of the through-space and through-bridge communications proposed by Nalewajski within the framework of the Orbital Communication Theory is used to evaluate the correlation effect between electron populations of two chemical bonds. Proposed in this paper purely-probabilistic formalism defines the correlation between two chemical bonds as determined by the difference between appropriately normalized fourth-order joint probabilities corresponding to direct (through-space) and indirect (through-bridge) communication within a four-state time-homogeneous Markov chain. Alternative bond-correlation coefficient introduced earlier by Yamasaki and Goddard is based on the concept of the hierarchy of statistical covariance operators. The proposed correlation coefficient requires computing only the second-order probability terms when working within the condensed-atomic resolution and thus they are far less computationally expensive making them suitable for correlation analysis of chemical interactions between large molecular fragments.

Ubiquitous Alkyne Activity

Chemistry Over the past decade, the coupling of terminal alkynes with azides—so efficient it's been termed a “click” reaction—has become a prevalent means of linking together larger molecular fragments. One advantage of the scheme was that the alkynes appeared to be otherwise inert in cellular environments, thereby enabling high specificity in the design of various biochemical probes. Two studies now report that terminal alkynes do in fact react with certain enzymes. Ekkebus et al. show that appending the alkyne to the C terminus of ubiquitin leads to inhibition of deubiquitinating enzymes. Crystallography revealed covalent linkage to an active site cysteine, the product of formal Markovnikov addition of the thiol group across the carbon-carbon triple bond (though no reaction was observed with free thiol). Sommer et al. observed the same reaction of C-terminally propargylated small ubiquitin-like modifier (SUMO) with its protease. Scavenger experiments in both studies disfavor a radical mechanism. Both studies leveraged the observation for selective labeling of lysates and suggest that this reaction could have broad application in selective inhibition. J. Am. Chem. Soc. 135 , 2867; Bioorg. Med. Chem. 21 , 10.1016/j.bmc.2013.02.039 (2013).

Formation of a metal-to-nitrogen bond of normal length by a neutral sufonamide group within a tridentate ligand. A new approach to radiopharmaceutical bioconjugation.

We demonstrate that a tertiary sulfonamide group, N(SO2R)R'2, can rehybridize to form a M-N bond of normal length even when the group is in a linear tridentate ligand, such as in the new tridentate N(SO2R)dpa ligands derived from di-(2-picolyl)amine (N(H)dpa). N(SO2R)dpa ligands were used to prepare fac-[Re(CO)3(N(SO2R)dpa)](PF6 or BF4) complexes. Structural characterization of the new complexes established that the tertiary sulfonamide nitrogen atom binds to Re with concomitant sp(2)-to-sp(3) rehybridization, facilitating facial coordination. The new fac-[Re(CO)3(N(SO2R)dpa)]X structures provide the only examples for any metal with the sulfonamide as part of a noncyclic linear tridentate ligand and with a normal metal-to-nitrogen(tertiary sulfonamide) bond length. Rare previous examples of such normal M-N bonds have been found only in more constrained situations, such as with tripodal tetradentate ligands. Our long-term objectives for the new tridentate N(SO2R)dpa ligands are to develop the fundamental chemistry relevant to the eventual use of the fac-[M(I)(CO)3](+) core (M = (99m)Tc, (186/188)Re) in imaging and therapy. The sulfonamide group uniquely contributes to two of our goals: expanding ways to conjugate the fac-[M(I)(CO)3](+) core to biological molecules and also developing new symmetrical tridentate ligands that can coordinate facially to this core. Tests of our conjugation method, conducted by linking the fac-[Re(I)(CO)3](+) core to a new tetraarylporphyrin (T(N(SO2C6H4)dpa)P) as well as to a dansyl (5-(dimethylamino)naphthalene-1-sulfonyl) group, demonstrate that large molecular fragments can be tethered via a coordinated tertiary sulfonamide linkage to this core.

Microscale affinity purification of trypsin reduces background peptides in matrix-assisted laser desorption/ionization mass spectrometry of protein digests

The use of trypsin for protein digestion is hampered by its autolysis and low thermostability. Chemical modifications have been employed to stabilize the enzyme. Modified trypsin (e.g. methylated) usually enables performing digestions at elevated temperatures, but it still produces autolytic peptides. In this work, unmodified bovine trypsin was subjected to a microscale affinity chromatography on Arginine Sepharose (ASE) or Benzamidine Sepharose (BSE), which utilized the principle of active-site ligand binding. Trypsin was retained on the sorbents in ammonium bicarbonate as a binding buffer. After washings to remove unbound impurities, the enzyme was eluted by arginine as a free ligand (from ASE) or by diluted hydrochloric acid (from BSE). MALDI-TOF mass spectrometry confirmed removal of large molecular fragments as well as autolytic and other background peptides. Consequently, the purified trypsin was tested for its performance in procedures of in-gel digestion of protein standards and selected urinary proteins from real samples. It has been shown that the affinity purification of trypsin decreases significantly the number of unmatched peptides in peptide mass fingerprints. The presence of arginine in the digestion buffer was found to reduce intensity of autolytic peptides. As a result, the described purification procedure is applicable in a common proteomic routine.

Individual identification by DNA polymorphism using formalin‐fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin-fixed material is very limited. In the present study the use of 6 week formalin-fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin-fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin-fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide-primed PCR (DOP-PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP-PCR products. Furthermore, large molecular size fragments were purified from the DOP-PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.

Four-Photon Laser Spectroscopy of Liquid Water and Aqueous Solutions of α-Chymotrypsin in the Range ±5 cm[sup –1

The resonances located at 1.2, 1.6, 2.0, and 2.3 cm−1 with a width of ∼0.2 cm−1 were observed for the first time in the range ±5 cm−1 of the four-photon Rayleigh wing spectra of distilled water and aqueous solutions of the protein α-chymotrypsin. The line at 2.3 cm−1 belongs to the rotational transition 3(2, 1)–4(1, 4) of the ground vibrational state of water. In the presence of the protein, the spectrum is modified by the appearance of new lines, located at 0.74, 2.8, and 3.2 cm−1. The modification of the spectrum observed is interpreted as a manifestation of low-frequency vibrations of large molecular fragments in aqueous protein solutions and as a result of the structuring of water in the vicinity of protein molecules.

Is there a weak H-bond --> LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.

Bond Length Dependence of Cluster Expansions of Pi-Electron Energy

There are two long established methods for representing the delocalized portion of the pi-electron energy of conjugated molecules in a simple local fashion. One, introduced by Hess and Schaad, can be used to provide a very accurate estimate of the energy of acyclic polyenes. Another, introduced originally by Herndon, has been successfully applied to the energy of aromatic ring systems. Both methods can be shown to be forms of cluster expansions, in which the energy of a molecule is expressed exactly as a sum of (successively smaller) contributions from successively larger molecular fragments. As such, it should in principle be possible to combine the methods to provide a consistent description of molecules of almost any structure. However past attempts to do so have yielded physically unreasonable results. It is shown that the key missing ingredient in these attempts is a failure to account for bond stretching and compression. When the energy changes associated with bond length changes are accounted for properly, the combined cluster expansion is quite successful in accounting for molecular pi electron energies in a very compact form.

In situ observation of particle-induced desorption from a self-assembled monolayer by laser-ionization mass spectrometry

We studied particle-stimulated desorption processes of highly ordered, self-assembled monolayers of biphenyl-based thiols covalently bound to Au/mica substrates with laser postionization in combination with mass spectrometry. Direct evidence was obtained that large molecular fragments are removed from these monolayers during impact of electrons with a kinetic energy of 1 keV. The damage that accumulates in the self-assembled monolayer with increasing electron dose was measured using ion-beam, sputter-initiated laser probing. Our results show that electron-induced desorption competes with the gradual erosion of the monolayer by the formation of a carbonaceous residual layer on the substrate.

Polycyclooctatetraene (polyacetylene) produced with a ruthenium olefin metathesis catalyst

The area of highly conjugated organic polymers has commanded interest for quite some time. Polyacetylene (PA) is the (structurally) simplest conjugated organic polymer; however, its intractable nature has made its characterization quite difficult. The first successful PA film was produced in 1971 by Shirakawa and Ikeda from acetylene monomer and a highly concentrated Ziegler–Natta catalyst. Over the last 30 years, several methods have been introduced which allow for the synthesis of a pre-cursor polymer that can subsequently be transformed into PA. Unfortunately, many of these techniques either involve the extrusion of large molecular fragments that can limit the processing of these polymers or produce very sensitive and even explosive materials. Therefore, a forgiving and direct route to PA and substituted PA, such as ring opening metathesis polymerization (ROMP), which might lead to new substrates and amenable processing conditions is worth pursuing. The metathesis polymerization of 1,3,5,7-cyclooctatetraene (COT) has been reported previously, however, the only successful routes to date have focused on early transition metal catalysts (tungsten) which are sensitive to air, moisture, and functional groups. It would be advantageous to use a late transition metal catalyst (ruthenium) which is more tolerant towards air, moisture and functional groups. Unfortunately, the RuCl_2(☐CHPh)(PCy_3)_2, catalyst 1, was not able to polymerize COT presumably due to its lower activity. Here, we report the ROMP of COT by a highly active well defined ruthenium olefin metathesis catalyst, RuCl_2(☐CHPh)(PCy_3)(IMesH_2), catalyst 2, to produce films of polyacetylene (PA) with conductivities comparable to those first produced by Shirakawa.

A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.

DNA fragmentation characteristic of apoptosis and cell loss induced by kainic acid in rabbit retinas

We have examined whether in vivo exposure to the glutamate analogue, kainic acid, induces cell loss through apoptosis and/or through necrosis. The vulnerability of rabbit retinal cells was evaluated by routine histopathology. The DNA fragmentation was examined using an in situ method (TUNEL: TdT-mediated biotin-dUTP nick-end labelling) and agarose gel electrophoresis of extracted retinal DNA. Retinas were examined at 30 min, and 4, 16, 24 and 36 h, and 2–5 days following the intraocular administration of 140 nmol kainic acid. Although pyknotic cells could be seen already at 30 min post-injection, TUNEL-labelled nuclei were first observed 4 h after the injection. A relatively large number of pyknotic cells and of TUNEL-labelled nuclei were still seen at 5 days post-injection. Pyknotic cells were seen throughout the inner nuclear layer (mostly in the proximal half of the layer) and in the ganglion cell layer. The TUNEL-labelled nuclei were almost only seen in the proximal inner nuclear layer. Analysis of DNA by electrophoresis revealed the presence of large molecular weight fragments 4 h after the injection, and of oligonucleosome-size fragments between 16 h and 2 days after the injection. The present study thus presents evidence that, in our model, the retinal cell loss induced by kainic acid is preceded, probably in most cells, by a fragmentation of DNA characteristic of apoptotic cell death. The process of cell loss following kainic acid administration was found to be relatively slow, further suggesting that a programmed type of cell death, which eventually induces apoptosis, is involved. No indication that cells were lost also through necrosis was obtained.

Endo-[beta]-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence.

Lettuce (Lactuca sativa L.) endosperm cell walls isolated prior to radicle emergence underwent autohydrolysis, the rate of which was correlated with whether radicle emergence would subsequently occur. Extraction of endosperm cell walls with 6 M LiCl suppressed autohydrolysis, and the desalted extract possessed activity that was capable of hydrolyzing purified locust bean galactomannan but not arabinogalactan, carboxymethylcellulose, glucomannan, polygalacturonic acid, tomato galactomannan, or native lettuce endosperm cell walls. Some hydrolytic activity was detected on endosperm cell walls if they were modified by partial trifluoroacetic acid hydrolysis or pretreatment with guanidinium thiocyanate. In extended incubations the cell wall enzyme extract released only large molecular mass fragments from locust bean galactomannan, indicating primarily endo-activity. Galactomannan-hydrolyzing activity in the cell wall extract increased as a function of imbibition time and was greatest just prior to radicle emergence. Thermoinhibition (imbibition at 32[deg]C) or treatment with abscisic acid at a temperature optimal for germination (25[deg]C) suppressed both germination and endosperm cell wall mannanase activity, whereas alleviation of thermoinhibition with gibberellic acid was accompanied by significant enhancement of mannanase activity. We conclude that a cell wall-bound endo-[beta]-mannanase is expressed in lettuce endosperm prior to radicle emergence and is regulated by the same conditions that govern germination.

About some pecularities of ionization in radio-frequency glow discharge (rfGD).

Two modes of rfGD formation were observed: intense and mild. In the mild mode mainly molecular peaks and large molecular fragments were observed and they were more abundant than with electron impact ionization. In the intense mode more molecular destruction was observed.